An Incompatibility between a mitochondrial tRNA and its nuclear-encoded tRNA synthetase compromises development and fitness in Drosophila

Abstract

Mitochondrial transcription, translation, and respiration require interactions between genes encoded in two distinct genomes, generating the potential for mutations in nuclear and mitochondrial genomes to interact epistatically and cause incompatibilities that decrease fitness. Mitochondrial-nuclear epistasis for fitness has been documented within and between populations and species of diverse taxa, but rarely has the genetic or mechanistic basis of these mitochondrial-nuclear interactions been elucidated, limiting our understanding of which genes harbor variants causing mitochondrial-nuclear disruption and of the pathways and processes that are impacted by mitochondrial-nuclear coevolution. Here we identify an amino acid polymorphism in the Drosophila melanogaster nuclear-encoded mitochondrial tyrosyl-tRNA synthetase that interacts epistatically with a polymorphism in the D. simulans mitochondrial-encoded tRNA(Tyr) to significantly delay development, compromise bristle formation, and decrease fecundity. The incompatible genotype specifically decreases the activities of oxidative phosphorylation complexes I, III, and IV that contain mitochondrial-encoded subunits. Combined with the identity of the interacting alleles, this pattern indicates that mitochondrial protein translation is affected by this interaction. Our findings suggest that interactions between mitochondrial tRNAs and their nuclear-encoded tRNA synthetases may be targets of compensatory molecular evolution. Human mitochondrial diseases are often genetically complex and variable in penetrance, and the mitochondrial-nuclear interaction we document provides a plausible mechanism to explain this complexity.

Figure 1. Effects of a mitochondrial–nuclear interaction on development.

(A) Mitochondrial-nuclear interactions between the D. simulans simw⁵⁰¹ mtDNA and the D. melanogaster OreR nuclear genome significantly extend egg-to-adult development time in both sexes (mtDNA×nuclear interaction: P_ANOVA≤0.0001, Table S1). The D. simulans sm21 mtDNA is closely related to simw⁵⁰¹, but has no effect on development time relative to the D. melanogaster ore mtDNA. (B and C) Both larval development and metamorphosis are delayed in (simw⁵⁰¹);OreR. Crosses between mitochondrial-nuclear genotypes indicated that the OreR nuclear effect on development is similar in males and females, autosomal and largely recessive (h = 0.18, 0.19 and 0.23, for pupation time, and male and female eclosion times, respectively, where h = 0 is complete dominance of AutW132 and h = 0.5 is additivity). Listed below the graphs are the (mtDNA);sex chromosome;autosome genotypes (O = OreR, A = AutW132). O/A and A/O heterozygotes indicate the offspring of reciprocal crosses and differ in the parent-of-origin of the autosomes (maternal/paternal). The difference in time from egg to pupation between (simw⁵⁰¹);AutW132 and (simw⁵⁰¹);OreR is approximately 65 hours. The difference in time from egg to adult emergence between (simw⁵⁰¹);AutW132 and (simw⁵⁰¹);OreR is 80 and 82 hours in males and females, respectively. The 65 hour delay in larval development and the additional 15–17 hour delay during metamorphosis between (simw⁵⁰¹);AutW132 and (simw⁵⁰¹);OreR are both statistically significant (P _t-test<0.001).

Figure 2. Effects of a mitochondrial–nuclear interaction on adult fecundity and sensory structures.

(A) The simw⁵⁰¹ mtDNA decreases the total number of eggs females laid by 50% only in the OreR nuclear background (mtDNA×nuclear interaction: F = 9.772, P = 0.004, N = 6–11 females per genotype). There is a main effect of the nuclear genome on fecundity, presumably because OreR and AutW132 are from different populations and differ at thousands of loci across their genomes. (B) A second experiment reveals the same significant mitochondrial-nuclear interaction (P_ANOVA = 0.001, Table S1) and also shows that the closely related D. simulans mtDNA sm21 does not decrease fecundity in either nuclear background. (C) The simw⁵⁰¹ mtDNA shortens adult mechanosensory bristles by 50% in the OreR nuclear background. (D) Measurement of the posterior scutellar bristles reveals a significant mitochondrial-nuclear interaction effect on bristle length (P_ANOVA≤0.001, Table S1). There was no sex-by-genotype interaction, and sexes are pooled in this plot. Some error bars are smaller than the symbols.

Figure 3. A mtDNA polymorphism in the D. simulans mt-tRNA^Tyr anticodon stem.

The D. simulans simw⁵⁰¹ tRNA^Tyr has a G∶C to G∶U mutation in the anticodon stem relative to the D. simulans sm21 and D. melanogaster mtDNAs. Shown is the D. simulans sm21 sequence.

Figure 4. Genetic mapping implicates an interaction between the mt-tRNA^Tyr and its nuclear-encoded mt-TyrRS.

(A) Chromosome segregation mapping using dominant, visible markers on the second and third chromosomes (Cy and Sb, respectively) revealed that the developmental delay is caused by a largely recessive factor on the OreR second chromosome. Only flies homozygous for the OreR second chromosome (squares) have extended development time. (B) Meiotic mapping using visible markers on the second chromosome indicates that flies with the simw⁵⁰¹ mtDNA that are homozygous for the OreR second chromosome (red) at the marker speck (sp) take significantly longer to develop than flies with the simw⁵⁰¹ mtDNA that are heterozygous for OreR and the mapping chromosome allele at this marker (gray), resulting in a significant marker-trait association (LOD score). (C) Two overlapping chromosomal deficiencies (BSC606 and BSC856) at the tip of Chromosome arm 2R fail to complement the OreR nuclear factor and significantly extend development time when combined with the simw⁵⁰¹ mtDNA (P_ANOVA<0.0001, both deficiencies). Two neighboring deficiencies (BSC780 and ED4061) complement the nuclear factor and restore development time to that of controls. Gray bars represent mean development time of (simw⁵⁰¹);OreR individuals inheriting the deficiency chromosome and white bars are control siblings inheriting a compatible balancer chromosome. The effects of these deficiencies are independent of sex, require the simw⁵⁰¹ mtDNA (Figure S2), and implicate Aatm, the only gene contained in both BSC606 and BSC856 with annotated mitochondrial function.

Figure 5. An amino acid change in the mt-TyrRS gene Aatm is incompatible with the mt-tRNA^Tyr polymorphism.

(A) A nonsynonymous SNP in the D. melanogaster OreR nuclear-encoded mt-TyrRS gene Aatm changes a highly conserved alanine to a valine at a residue adjacent to the class I aaRS “KMSKS” signature sequence located in a loop that connects the catalytic domain to the anticodon binding (ACB) domain . Nine Drosophilid species sequences are followed by mosquito (Anopheles gambiae), zebrafish (Danio rerio), mouse (Mus musculus) and human. (B) Both the transgenic Aatm^Aut allele and the Aatm^Ore_V275A allele that reverts the valine in the OreR allele to the conserved alanine significantly recover development time in a simw⁵⁰¹ mitochondrial background and recapitulate the developmental difference between the (simw⁵⁰¹);OreR and (simw⁵⁰¹);AutW132 genotypes. Boxplots show the distributions of mean development time for all individuals emerging from a single vial, and notches indicate the approximate 95% confidence intervals around the medians. Numbers above the boxes indicate the magnitude of the reduction in development time in days, relative to the incompatible Aatm^Ore allele (P_Tukey<0.0001, both alleles). Normalized development time is relative to control siblings that emerge from the same vial. Data were pooled across sexes, as the effects were the same in males and females (Figure S3 and Table S2).

Figure 6. The mitochondrial-nuclear incompatibility decreases the activity of OXPHOS complexes that are mitochondrially translated.

(simw⁵⁰¹);OreR individuals have significantly reduced activities of OXPHOS complexes I, III and IV, which contain subunits encoded in the mtDNA and translated in mitochondria (mtDNA×nuclear, P_ANOVA<0.01, each complex). In contrast, the activities of complex II and citrate synthase, which function in the mitochondria but are encoded entirely by nuclear genes and translated in the cytoplasm, are similar across genotypes (mtDNA×nuclear, P_ANOVA>0.20, both activities). Plots combine male and female data, as the mitochondrial-nuclear interaction effects were the same in both sexes (Table S3).